DNA sequences encoding growth/differentiation

Details DNA sequences encoding growth/differentiation DNA sequences encoding growth/differentiation

1998-04-03

2001-03-06

Mertz, Prema (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S071100, C435S071200, C435S471000, C435S320100, C435S325000, C435S252300, C435S254100, C435S254110

Reexamination Certificate

active

06197550

ABSTRACT:

The present invention relates to DNA sequences encoding novel growth/differentiation factors of the TGF-&bgr; family. In particular, it relates to novel DNA sequences encoding TGF-&bgr;-like proteins, to the isolation of said DNA sequences, to expression plasmids containing said DNA, to microorganisms transformed by said expression plasmid, to the production of said protein by culturing said transformant, and to pharmaceutical compositions containing said protein. The TGF-&bgr; family of growth factors comprising BMP, TGF, and Inhibin related proteins (Roberts and Sporn, Handbook of Experimental Pharmacology 95 (1990), 419-472) is of particular relevance in a wide range of medical treatments and applications. These factors are useful in processes relating to wound healing and tissue repair. Furthermore, several members of the TGF-&bgr; family are tissue inductive, especially osteo-inductive, and consequently play a crucial role in inducing cartilage and bone development.
Wozney, Progress in Growth Factor Research 1 (1989), 267-280 and Vale et al., Handbook of Experimental Pharmacology 95 (1990), 211-248 describe different growth factors such as those relating to the BMP (bone morphogenetic proteins) and the Inhibin group. The members of these groups share significant structural similarity. The precursor of the protein is composed of an aminoterminal signal sequence, a propeptide and a carboxyterminal sequence of about 110 amino acids, which is subsequently cleaved from the precursor and represents the mature protein. Furthermore, their members are defined by virtue of amino acid sequence homology. The mature protein contains the most conserved sequences, especially seven cysteine residues which are conserved among the family members. The TGF-&bgr;-like proteins are multifunctional, hormonally active growth factors. They also share related biological activities such as chemotactic attraction of cells, promoting cell differentiation and their tissue-inducing capacity, such as cartilage- and bone-inducing capacity. U.S. Pat. No. 5,013,649 discloses DNA sequences encoding osteo-inductive proteins termed BMP-2 proteins (bone morphogenetic protein), and U.S. patent applications Ser. Nos. 179 101 and 179 197 disclose the BMP proteins BMP-1 and BMP-3. Furthermore, many cell types are able to synthesize TGF-&bgr;-like proteins and virtually all cells possess TGF-&bgr; receptors.
Taken together, these proteins show differences in their structure, leading to considerable variation in their detailed biological function. Furthermore, they are found in a wide variety of different tissues and developmental stages. Consequently, they might possess differences concerning their function in detail, for instance the required cellular physiological environment, their lifespan, their targets, their requirement for accessory factors, and their resistance to degradation. Thus, although numerous proteins exhibiting tissue-inductive, especially osteo-inductive potential are described, their natural role in the organism and, more importantly, their medical relevance must still be elucidated in detail. The occurrence of still-unknown members of the TGF-&bgr; family relevant for osteogenesis or differentiation/induction of other tissues is strongly suspected. However, a major problem in the isolation of these new TGF-&bgr;-like proteins is that their functions cannot yet be described precisely enough for the design of a discriminative bioassay. On the other hand, the expected nucleotide sequence homology to known members of the family would be too low to allow for screening by classical nucleic acid hybridization techniques. Nevertheless, the further isolation and characterization of new TGF-&bgr;-like proteins is urgently needed in order to get hold of the whole set of induction and differentiation proteins meeting all desired medical requirements. These factors might find useful medical applications in defect healing and treatments of degenerative disorders of bone and/or other tissues like, for example, kidney and liver.
Thus, the technical problem underlying the present invention essentially is to provide DNA sequences coding for new members of the TGF-&bgr; protein family having mitogenic and/or differentiation-inductive, e.g. osteo-inductive potential.
The solution to the above technical problem is achieved by providing the embodiments characterized in claims
1
to
14
. Other features and advantages of the invention will be apparent from the description of the preferred embodiments and the drawings. The sequence listings and drawings will now briefly be described.
SEO ID NO. 1 shows the nucleotide sequence of MP-52, i.e. the embryo derived sequence corresponding to the mature peptide and most of the sequence coding for the propeptide of MP-52.
Some of the propeptide sequence at the 5′-end of MP-52 has not been characterized so far.
SEO ID NO. 2 shows the nucleotide sequence of MP-121, i.e. the liver derived sequence corresponding to the mature peptide, the sequence coding for the propeptide of MP-121, and sequences 5′ and 3′ to the coding region. The start codon begins with nucleotide 128 of SEQ ID NO.2. The sequence coding for the mature MP121 polypeptide begins with nucleotide 836 of SEQ ID NO. 2. The stop codon begins with nucleotide 1184 of SEQ ID NO. 2. The sequence coding for the precursor protein has a length of 1056 bp. The sequence coding for the propeptide has a length of 708 bp and the sequence coding for the mature peptide has a length of 348 bp.
SEO ID NO. 3 shows the amino acid sequence of MP-52 as deduced from SEQ ID NO. 1.
SEQ ID NO. 4 shows the amino acid sequence of MP-121 as deduced from sequence SEQ ID NO.2. The sequence of the mature polypeptide begins with amino acid 237 of SEQ ID NO. 4. The precursor protein has a length of 352 amino acids. The propeptide and the mature peptide have a length of 236 and 116 amino acids, respectively.
SEO ID NO. 5 shows a part of the nucleotide sequence of the liver derived sequence of MP-121.
SEO ID NO. 6 shows a part of the nucleotide sequence of the embryo derived sequence of MP-52.
The shorter DNA-sequences SEQ ID NO. 5 and 6 can be useful for example for isolation of further members of the TGF-&bgr;-protein family.


REFERENCES:
patent: 0 222 491 (1996-10-01), None
patent: PCT/EP95/02552 (1995-11-01), None
patent: 93/16099 (1993-08-01), None
Hötten et al., “Cloning of a New Member of the TGF-&bgr; Family: A Putative New Activin &bgr;c Chain”, Biochem. & Biophys. Res. Comm., vol. 206, No. 2, 1995.
Chang et al. 1994) J. Biol. Chem. vol. 269, No. 45, pp. 28227-28234.
Rieger et al. Glossary of Genetics & Cytogenetics, pp. 17-19 Springer-Verlag, 4th Edition, 1976.
Ngo et al. 1994 Ch. 14, pp. 491-495 in The Protein Folding Problem & Tertiary Structure Prediction, K. Merz, Jr & S. Le Grand Editors, Birkhaüuser Boston.
Cunningham et al. (1989) Science vol. 244, pp. 1081-1095.
George et al. Ch 12 pp. 127-149 in “Macromolecular Sequencing & Synthesis”, Alan R. Liss., N.Y. (1988).

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